OBJECTIVE:
To evaluate the clinical value of lactate measurement and nucleated red blood
cell (NRBC) counts when compared to base excess (BE) in the blood collected
from the placental segment of the umbilical vein.METHODS:
25 umbilical cords from premature babies were sampled after placental delivery
and cord clamping. Babies were followed until discharge. Statistics involved
linear regression, Spearman's correlation, ROC curves, and Fisher's exact test.RESULTS:
The relationship between lactate in the umbilical vein blood and pH and BE was
significant (p < 0.0001). A 4.04 mmol/L lactate level showed a sensitivity
of 62.5% and a specificity of 94.1% in detecting pH <7.2 and BE < -10
mmol/L. NRBC counts were related to BE (p = 0.0095), but with a sensitivity
of 37.5% and specificity of 82.4% in detecting BE < -10 mmol/L.CONCLUSIONS:
Lactate is a valuable marker of fetal hypoxia when sampled from placental segment
veins. NRBC counts demonstrated low sensitivity for the detection of acidosis.

Keywords:
Lactate, erythroblasts, premature babies, fetal distress.

Introduction

Biochemical markers
are valuable tools for the detection of hypoxic-ischemic events. Historically,
fetal umbilical pH and base excess (BE) have been used as markers of hypoxic-ischemic
insult, reflecting the cellular production of metabolic acids in consequence
of hypoxemia. Lactate has been proposed as a useful method for the detection
of fetal hypoxia.1 Nucleated red blood cell (NRBC) counts are increased
in association with higher levels of erythropoietin in response to tissue hypoxia,
and studies have demonstrated the relationship between this marker and perinatal
hypoxia-ischemia.2,3 Both lactate and NRBC counts have been investigated
as predictors of short and long-term prognosis, with conflicting results.1,4-6

The aim of this
study was to evaluate lactate levels and NRBC counts in the blood from the placental
segment of the umbilical vein of premature babies in relation to BE values.
We also investigated correlations of lactate and nucleated red blood cells to
Apgar scores and maternal complications, and prospectively followed these babies
until hospital discharge, to assess the prognostic value of the markers with
respect to the short-term outcome.

Methods

Twenty-five premature
newborns, born at a tertiary hospital in Brazil, from April 2004 to January
2005, were assessed. Inclusion criteria were gestational age < 37 weeks,
weight < 2,000 g, and a written informed consent signed by the mother. The
study was approved by the local Research Ethics Committee. Congenital malformations,
maternal diabetes and blood group incompatibility were exclusion criteria. Immediately
after placental delivery, blood was sampled (0.5 mL) from the umbilical vein
in the segment close to the placenta, using 5-mL heparinized plastic syringes,
and then placed on ice. Blood was also sampled into sodium fluoride vials for
lactate measurements, and into K3-EDTA vials for blood cell counts. Analysis
of blood gases was performed within 30 minutes after sampling, using a Rapid
Lab 865 (Bayer) analyzer. Plasma lactate was analyzed by an enzymatic method
(Ortho-Clinical Diagnostics  Vitros 750, Johnson & Johnson). NRBC and leukocytes
were counted through manual microscopy of the blood smear, and confirmed by
a second technician. NRBC were calculated as the number of NRBC per 100 leukocytes.

Classically, acidosis
at birth has been defined as BE in umbilical arterial blood lower than -12 mmol/L,
which is approximately 2 standard deviations (SD) below the mean for normal
newborns.7-9 Nodwell et al. demonstrated that values of blood gases
are different in placental and umbilical segments after cord clamping and placental
delivery. The values observed for BE in the placental segment were -3.4 (mean)
± 2.2 (SD).10 We defined our endpoint for acidosis as BE equal
to or lower than -10 mmol/L, which corresponds to 3 SD below the mean, based
on these values.

For the short-term
outcome we prospectively collected information about the occurrence of perinatal
infection (defined by clinical signs and symptoms or positive blood culture,
or both), periventricular or intraventricular hemorrhage (assessed by cranial
ultrasound), necrotizing enterocolitis, need for vasoactive drugs, parenteral
nutrition, blood products, oxygen, mechanical ventilation (invasive or non-invasive).
Clinical information was also collected to calculate the Clinical Risk Index
for Babies (CRIB) score in the first 12 hours of life.11 Lactate
levels and NRBC correlations were investigated for each one of these factors.
Apgar scores and maternal complications (infection, hypertensive disease, premature
rupture of membranes more than 18 hours before birth) were also analyzed for
relationships with the markers. Maternal infection was defined as the presence
of fever, leukocytosis, or use of antibiotics for reasons such as urinary tract
infection or pneumonia at birth. As neurological examination of premature babies
is uncertain, this criterion was not included.

Statistical significance
of correlations between investigated markers and pH, BE, PCO2, O2
saturation and oxygen blood content was determined by linear regression analysis
with 99% confidence intervals. For comparison between scores (Apgar and CRIB)
and the markers, Spearman's non-parametric correlation was used. Through receiver
operating characteristic (ROC) curves and contingency tables, we determined
the value of the markers for identifying acidosis, and established the cutoff
values for this parameter. We also tested the presence of a value higher than
the defined cutoff values for the occurrence of perinatal outcome factors with
Fisher's exact test. The statistical analysis was performed using the Analyse-it
software (www.analyse-it.com).

Results

Clinical characteristics,
perinatal events and their relationships to the investigated markers are shown
in Table 1. Means and standard deviations were
determined as follows: lactate 4.26 ± 3.78 mmol/L; NRBC counts 20.52
± 37.96 cells/100 leukocytes; BE, -9.35 ± 5.99 mmol/L; pH, 7.22
± 0.17;CO2, 46.06 ± 17.74 mmHg. Figure
1 shows the correlation between umbilical vein blood values of lactate and
BE (R2 = 0.72, p < 0.0001). For the identification of acidosis
(BE < -10 mmol/L), the area under the ROC curve was 0.842. The sensitivity
of a lactate cutoff level equal to 4.04 mmol/L in relation to BE lower than
or equal to -10 mmol/L was 62.5%, and specificity was 94.1%, with a positive
predictive value equal to 83.3%, and negative predictive value of 84.2%. The
same values were observed for pH. Although pH and CO2 were not endpoints,
because of their variability caused by placental metabolism and gas exchange
after cord clamping, we observed a strong relationship between lactate and pH
(R2 = 0.82, p < 0.0001), and between lactate and PCO2
(R2= 0.6, p < 0.0001). There was a weak correlation between lactate
and bicarbonate (R2 = 0.23, p = 0.014), and no correlation was found
between lactate and O2 blood content, PO2 and hemoglobin
oxygen saturation, and Apgar scores. Apgar scores were also not related to pH
and BE.

NRBC counts were
related to BE (R2= 0.26, p = 0.009) and to pH (R2= 0.38,
p = 0.009). A cutoff value of 10 NRBC/100 leukocytes showed poor sensitivity
(40%) and specificity of 80% for more severe acidosis (BE lower than -12 mmol/L).
For our endpoint (BE lower than -10 mmol/L), NRBC count was not an adequate
test, with an area under the ROC curve equal to 0.577 (p = 0.26), sensitivity
of 37.5% and specificity of 82.4%, positive predictive value equal to 50% and
negative predictive value equal to 73.6%. The area under the ROC curve was 0.87
for the determination of pH < 7.2, for the cutoff value of NRBC, with sensitivity
of 50% and specificity of 88.2%. NRBC were not correlated to blood oxygen content
and PO2 and showed a weak correlation to oxygen saturation (R2
= 0.07, p = 0.018) and PCO2 (R2 = 0.33, p = 0.0027). NRBC
count and lactate levels were related to each other (R2 = 0.4, p
= 0.0008), and NRBC count also showed weak correlation to Apgar scores (p =
0.03 at 1 minute and p = 0.02 at 5 minutes).

Both lactate levels
and NRBC count were unable to predict short-term perinatal complications, with
an exception: from the seven babies with NRBC counts equal to or higher than
10/100 leukocytes, three developed necrotizing enterocolitis (42%, p = 0.026),
with one intestinal perforation. Lactate levels and NRBC counts were not related
to maternal complications.

Discussion

The diagnosis of
hypoxia-ischemia at birth is critical, and combining clinical markers with laboratory
data would be useful to identify premature infants with higher risk for neurological
damage.4

Samples from the
umbilical artery may be limited by small volume and by the difficulty in collecting
them, especially in thin umbilical cords. The umbilical vein, close to the site
of placental insertion, can provide an alternative source for sampling, but
only after placental delivery. Placental metabolism and gas exchange proceed
after delivery and cord clamping, affecting measurements in vein blood. This
is particularly true for oxygen measurements and PCO2, but Nodwel
et al. demonstrated that the agreement between BE in blood from the placental
segment of the umbilical vein, and in blood from the umbilical segment of the
umbilical vein or artery, is acceptable for clinical purposes.10
We observed that in blood from the placental segment of the umbilical vein after
placental delivery, lactate maintains the same good correlation to BE previously
reported for arterial measurements.1 Thus, lactate has a potential
as a simple and inexpensive tool for the diagnosis of metabolic acidosis associated
with hypoxia-ischemia in premature infants. A whole blood gas analysis costs
nearly three times more than a single lactate measurement.

Elevated NRBC counts
in umbilical venous blood have been correlated to acute and chronic antepartum
asphyxia.12 Maternal nucleated blood cells can transfer into fetal
circulation when uteroplacental perfusion is impaired, but this is not significant
for counts (one maternal cell to at least 100 fetal cells).13 NRBC
counts in placental circulation are also correlated to counts in umbilical blood.14
These facts, coupled with the assumption that NRBC count in umbilical vein blood
is not influenced by placental metabolism and cord clamping, make it attractive
as one more tool for monitoring the condition of premature newborns. We found
that NRBC counts are weakly related to BE and pH, with low sensitivity and positive
predictive values. NRBC count cannot be recommended for routine clinical diagnosis
of perinatal acidosis, since classical markers and lactate measurements are
more adequate.

Neither lactate
nor NRBC counts were useful to predict the short-term outcome as single markers.
Cases of necrotizing enterocolitis with elevated NRBC counts have been previously
reported,15 suggesting that NRBC counts in premature infants can
help to identify risk groups. More prospective studies are needed.